Supplementary Material

Plant growth promoting rhizobacteria Dietzia natronolimnaea modulates the expression of

stress responsive genes providing protection of wheat from salinity stress

Nidhi Bharti, Shiv Shanker Pandey, Deepti Barnawal, Vikas Kumar Patel, Alok Kalra

Supplementary Table S1 Effect of STR1 inoculation on fresh root weight, dry weight and

root length of wheat plants grown under non-saline and saline conditions

Treatments Fresh root weight(g)

Dry root weight(g)

Root length(cm)

Control 0.0742ab 0.0190b 16.10bc

Control+Salt 0.0536c 0.0153d 14.67c

STR1 0.0785a 0.0217a 18.67a

STR1+Salt 0.0691b 0.0175c 17.50ab

Wheat plants were grown hydroponically in Hoagland nutrient solution for 12 d. Sodium

chloride was added to the nutrient solution to obtain a final concentration of 100 mM . Values

are mean of ten replicates ± standard error of means. Means, followed by different letters in

the same column are statistically different according to Duncan’s multiple range test

(P<0.05). Control=without bacterial treatment; STR1 = plants inoculated with Dietzia

natronolimnaea

Supplementary Table S2 List of primers used for PCR

Gene

amplified

Primer

name

Sequence Reference

16S rRNA fD1 5΄-AGAGTTTGATCCTGGCTCAG-3΄ Weisburg et al. 1991

rP2 5΄-ACGGCTACCTTGTTACGACTT-3΄

nifH Pol F 5´-TGCGAYCCSAARGCBGACTC-3´ Poly et al. 2001

Pol R 5´-ATSGCCATCATYTCRCCGGA-3´

Supplementary Table S3 List of primers used for quantitative real time PCR analysis

*Primers for the TaNHX were designed from the highly conserved homologue regions of allavailable TaNHX isoforms (NCBI Accession Nos. AY040245, AY040246 and AY296910)from wheat (Saqib et al. 2005).#Primers were designed using Primer Express Software v2.0 (Applied Biosystems).

Gene Primer Sequence ReferenceTaWRKY10(EF368361)

Forward 5′-AGCTCGTCTGTGCAGTGCACTTAT-3′ Li et al. 2013Reverse 5′-TCGTGTACATGCATCCGTGAGATT-3′

TaWRKY17(JK546463)

Forward 5'-GACCAAGCGGCTCAACGAT-3' Garg et al. 2012Reverse 5'-GGTGCATATGCTAGCTTG-3'

TaSOS1(AY326952)

Forward 5'- GTTGTCGGTGAGGTCGGAGGG -3' Ramezani et al.2013Reverse 5'- TCATCTTCTCCTACCGCCCTGC-3'

TaSOS4(AY337321)

Forward 5'-ATCCAGTCCCACACCGTCCA -3'Reverse 5'- GCTGATTGCCATTGAGAACCTGTC-3'

TaST(EF675609)

Forward 5´-CGCAGGCCGTCGTCATG-3´ Huang et al.2012Reverse 5´-GACTGATCCTGCCAGCAAACAC-3'

POD(X56011)

Forward 5'-CAGCGACCTGCCAGGCTTTA-3' Jiang et al. 2012Reverse 5'-GTTGGCCCGGAGAGATGTGG-3'

MnSOD(GI1622928)

Forward 5'-CAGAGGGTGCTGCTTTACAA-3' Baek andSkinner 2003Reverse 5'-GGTCACAAGAGGGTCCTGAT-3'

CAT(GI5711144)

Forward 5'-CCATGAGATCAAGGCCATCT-3'Reverse 5'-ATCTTACATGCTCGGCTTGG-3'

APX(TC22268)

Forward 5'-GCAGCTGCTGAAGGAGAAGT-3'Reverse 5'-CACTGGGGCCACTCACTAAT-3'

GPX(TC22467)

Forward 5'-CCCCCTGTACAAGTTCCTGA-3'Reverse 5'-GTCAACAACGTGACCCTCCT-3'

GR(TC84151)

Forward 5'-TGCGTCCCGAAGAAGATACT-3'Reverse 5'-GTTGATGTCCCCGTTGATCT-3'

TaNHX* Forward 5'-ATCTACYTSCTCCCKCCCAT-3' Saqib et al. 2005Reverse 5'-AGATAGCYCCAATTGCAAGA-3'

TaMYB33(JN584645)

Forward 5'-GTCGTCCGCCACAGATTACTC-3' Qin et al. 2012Reverse 5'-CGAACGATTATTGTTCCCTTCAC-3'

TaOPR1(JQ409278)

Forward 5'-ACTGCCACGACTCCGACCC-3' Dong et al. 2013Reverse 5'-CCGATGCGACCGCCTTG-3'

Actin(AB181991)

Forward 5'-CGAAACCTTCAGTTGCCCAGCAAT-3'Reverse 5'-ACCATCACCAGAGTCGAGCACAAT-3'

HAK1(JF495466)

Forward 5’-ACGCTTACGGGATCTGTGTG-3’ This study#

Reverse 5’-GAGCCGAACACGACGTAGAA-3’hkt1

(U16709)Forward 5’-ATGGGCCGGGTGAAAAGATT-3’Reverse 5’-TCCAGAAGGGGTGAACATGC-3’

ABARE(JK546530)

Forward 5’-CGATGATGAACTCCACTCA-3’ Garg et al. 2012Reverse 5’-ATGATATAGTTCCAAAGG-3’

Supplementary Fig. S1: Neighbor-joining tree showing the phylogenetic relationship

between Dietzia natronolimnaea STR1 and reference strains.

The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree

with the sum of branch length = 0.29013879 is shown. The percentage of replicate trees in

which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown

next to the branches. The tree is drawn to scale, with branch lengths in the same units as those

of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances

were computed using the Tamura 3-parameter method and are in the units of the number of

base substitutions per site. The analysis involved 29 nucleotide sequences. Codon positions

included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were

eliminated. There were a total of 1395 positions in the final dataset. Evolutionary analyses

were conducted in MEGA5.

Supplementary Fig. S2: Growth curve for Dietzia natronolimnaea STR1 at different

conditions. (a) Growth pattern at different levels of NaCl viz., 0, 0.5 and 1 M NaCl (at

neutral pH). (b) Growth pattern at different pH levels viz., 3, 7 and 9.

Supplementary Fig. S3: Scanning electron microscopy images showing the colonization

of 12 days old wheat seedlings roots by Dietzia natronolimnaea STR1. (a) pure culture (b)

un-inoculated roots (c) inoculated roots. Arrows indicate bacteria.

Supplementary Fig. S4: Effect of STR1 inoculation on Catalase and Ascorbate

peroxidase activity of wheat plants under non-saline and saline conditions. Catalase and

Ascorbate peroxidase activity was measured in leaves of non-inoculated (control) and STR1-

inoculated 60 d old wheat plants grown in soil and supplemented with 150 mM NaCl.

Control: without bacterial treatment, STR1: plants inoculated with Dietzia natronolimnaea

STR1. Values are mean of five replicates ±standard error of means. Different letters indicate

statistically significant differences between treatments (Duncan’s multiple range test P<0.05)

Estimation of Catalase activity

Catalase (EC 1.11.1.6) activity was estimated according to Bergmeyer 1970 which

measures the initial rate of disappearance of H2O2 at 240 nm. 0.5 g tissue was ground in a

cold mortar and pestle using liquid nitrogen and suspended in 1.5 mL of homogenization

buffer solution (50 mM Tris-HCl, 0.1 mM EDTA, 0.2% TritonX-100, 1 mM PMSF, 2 mM

DTT). The suspension was centrifuged at 14,000 rpm for 30 min at 4 ºC. The supernatant was

taken for the enzyme assay.

Estimation of Ascorbate Peroxidase (APX) activity

APX (EC 1.11.1.11) activity was measured according to Nakano and Asada 1981.

The assay depends on the decrease in absorbance at 290 nm with ascorbate oxidation. 0.5 g

tissue, grinded in a cold mortar and pestle with liquid nitrogen and was suspended in 1.5 mL

homogenization buffer [50 mM Na-P Buffer (pH 7.0), 2% PVPP, 0.1 mM EDTA, 2 mM

ascorbate]. The suspension was centrifuged at 14,000 rpm for 30 min at 4 ºC and the

supernatant taken for the enzyme assay.

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